Leishmania parasite causes human disease (Leishmaniasis) with clinical symptoms ranging from self healing cutaneous lesions to fatal visceral infection. The lack of understanding of the mechanism by which Leishmania parasite causes disease poses a serious public health risk worldwide and in particular for U. S. military personnel their families and tourists either living or travelling in endemic areas. As a first step towards understanding the molecular mechanism of Leishmania pathogenesis, we have began to analyze the processes that are involved in parasite life cycle in transformation from an avirulent to virulent form. We have identified several differentially regulated genes of Leishmania donovani, a viscerotropic parasite, using various molecular biology techniques such as differential hybridization and arbitarily primed polymerase chain reaction (AP-PCR). We have characterized one such gene that codes for a homologue of a mammalian chaperone protein calretciculin (CR) which is differentially regulated. In addition,CR along with other chaperones has been shown to be important for glycosylation and intracellular transport of membrane and secretory proteins in the higher eukaroyotes. Very little is known about this process in parasites in general and Leishmania in particular. Further, membrane proteins or secretory proteins of Leishmania have been implied for its defense mechanism against killing by the host, we attempted to alter the expression of CR gene in L. donovani by gene-knock out so as to alter the biology of these proteins to create attenauted parasites. Deletion of CR gene was lethal for the parasite. Therefore, we attempted to alter the expression of endogenous CR by overexpressing complete coding region or portions of the coding region of CR. We obtained parasites which are overexpressing vaious regions of CR. Currently, we are analyzing the CR mutant parasites for their ability to alter the expression of Leishmanial membrane and secretory proteins. Such an analysis will allow us to test the potential of CR mutants as attenuated parasites for Leishmania vaccine.